HCV is one of the most prevalent causes of chronic liver disease in the United States, which accounts for about 15 percent of acute viral hepatitis, 60 to 70 percent of chronic hepatitis, and up to 50 percent of cirrhosis, end-stage liver disease, and liver cancer. Almost 4 million Americans, or 1.8 percent of the U.S. population, have antibodies to HCV (i.e., anti-HCV antibodies), indicating ongoing or previous infection with the virus. Hepatitis C causes an estimated 8,000 to 10,000 deaths annually in the United States. While the acute phase of HCV infection is usually associated with mild symptoms, some evidence suggests that only about 15% to about 20% of the infected people will clear HCV.
HCV is a small, enveloped, single-stranded positive strand RNA virus in the Flaviviridae family. The genome includes approximately 10,000 nucleotides and encodes a single polyprotein of about 3,000 amino acids. All of the protein products of HCV are produced by proteolytic cleavage of the polyprotein, carried out by one of three proteases: the host signal peptidase, the viral self-cleaving metalloproteinase (NS2), and the viral serine protease (NS3/4A). The combined action of these enzymes produces the structural proteins (C, E1 and E2) and non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) that are required for replication and packaging of the viral genomic RNA. NS5B is the viral RNA-dependent RNA polymerase (RDRP) that is responsible for the conversion of the input genomic RNA into a minus strand copy (complimentary RNA, or cRNA); the cRNA then serves as a template for transcription by NS5B of the positive sense genomic/messenger RNA. The HCV replicase is the complex of proteins that are necessary for the accurate and efficient synthesis of viral replicon RNA.
Currently, the only effective therapy against HCV is alpha-interferon, which reduces the amount of virus in the liver and blood (e.g., viral load) in only a small proportion of infected patients. Standard forms of interferon, however, are now being replaced by pegylated interferons (peginterferons), alpha interferons that have been modified chemically by the addition of a large inert molecule of polyethylene glycol. At the present time, the optimal regimen including interferon appears to be a 24- or 48-week course of a combination of pegylated alpha interferon and the nucleoside ribavirin, an oral antiviral agent that has activity against a broad range of viruses. Nonetheless, response rates to the combination interferon/ribavirin therapy may be moderate for certain HCV genotypes, i.e., a response rate of about 50% to about 60%, although response rates for selected genotypes of HCV (notably genotypes 2 and 3) are typically higher. Another drawback to the current therapy is that there are often significant adverse side effects associated with each of these agents including, for example, flu-like symptoms; bone marrow suppressive effects; neuropsychiatric effects such as marked irritability, anxiety, personality changes, depression, and even suicide or acute psychosis; histamine-like side effects; and anemia.
Taken together, the preceding facts indicate a significant need for effective small molecule inhibitors of HCV replication that do not suffer from the above-mentioned drawbacks. A particularly useful class of inhibitors of HCV, as well as other positive strand RNA viruses, is inhibitors of viral RNA synthesis.
While accurate and efficient assays for identifying HCV RNA synthesis inhibitors may be useful tools for identifying effective small molecule HCV therapeutics, no such system has been developed. An in vitro replication assay using recombinant NS5B polymerase has been reported. However, in this system, the purified form of NS5B polymerase lacked template specificity and produced various lengths of RNA products. These phenomena are very different from HCV RNA replication in vivo. To better reflect the HCV RNA replication process in the cell, a cell-free HCV replication system was established using whole cell lysates or membrane fractions of cells expressing the HCV replicon. In this cell-free system, radioactive P32-UTP or P32-CTP was used to label newly synthesized HCV RNA, and then the reaction products were resolved by gel electrophoresis, followed by autoradiography. Because these assays require gel electrophoresis to separate the full length HCV RNA from other RNA molecules, the results are difficult to quantify, often inaccurate, and poorly reproducible. Additionally, while it appears to be clear that RNA elongation occurs in this cell-free system, there is no convincing evidence that de novo RNA initiation occurs in this system.